The present invention relates to a process for preparing a highly purified high-melting crystalline form of chenodeoxycholic acid (=3.alpha.,7.alpha.-dihydroxy-5.beta.-cholanic acid).
Chenodeoxycholic acid, one of the major bile acids occurring in human bile and some animal biles, possesses valuable therapeutic properties, in that it is capable of reducing and/or dissolving cholesterol gall-stones in humans, and therefore is useful in the medical treatment of gall-stones and of metabolic disorders or diseases which lead to the formation of cholesterol gall-stones.
Chenodeoxycholic acid, which hereinafter will be abbreviated as CDCA, is known to exist in several polymorphic forms. The literature reports the existence of three crystalline forms having different melting points, and of at least one amorphous form of CDCA melting around 140.degree. C., and furthermore of crystalline CDCA/solvent complexes melting at about 120.degree. C. (see G. Giuseppetti et al, Il Farmaco, Ed.Sc. 33, 64, 1978). According to G. Giuseppetti et al, the crystalline polymorphic forms comprise a high-melting form having a melting point of about 168.degree. C., and two lower melting forms having a melting point of about 138.degree. C. and of about 119.degree. C. respectively.
The main natural sources of CDCA are animal biles. Various procedures for isolating CDCA as such from natural animal sources have been reported (see e.g., U.S. Pat. Nos. 3,931,256 and 3,919,266). Another important partially synthetic route for obtaining CDCA comprises synthesizing CDCA from cholic acid which in turn may be isolated from animal bile (see e.g., Fieser and Rajagopalan, J. Am. Chem. Soc. 72, 5530 (1950), and Hofmann, Acta Chem. Scand. 17, 173 (1963).
Since certain of the compounds which occur together with CDCA in the bile and/or are formed during the isolation and/or synthesis of CDCA (e.g., related bile acids and esters) are known to possess hepatotoxid properties, it is of utmost importance that for therapeutic purposes a form of CDCA is used which can easily and unambiguously be characterized by its physical properties, which can be provided in highly purified form, and wherein any, even minor, contamination with impurities, e.g., the above-mentioned related compounds and/or solvent residues, can be easily recognized and subsequently be removed.
Both, CDCA conventionally prepared by isolation from animal bile material or by synthesis from cholic acid, are usually obtained in a form melting around or below 140.degree. C. Various methods for purifying the raw product have been proposed, involving recrystallization of the CDCA from various solvent systems optionally combined with a chromatographic purification step and/or intermediate formation of salts or esters of the CDCA. Most of these methods result in purified products having a melting point around or slightly above 140.degree. C.
For example, in the process for isolating CDCA from animal bile disclosed in U.S. Pat. Nos. 3,931,256 and 3,919,266, CDCA is separated in form of its crude barium salt which is treated with ethylacetate and HCl to obtain a solution of CDCA in ethylacetate, from which CDCA is precipitated by addition of n-hexane, yielding a form of CDCA having a melting point of 140.degree.-142.degree. C. (see Example V). This product may be further purified by the following methods (Examples VI-VIII): countercurrent distribution of a solution in ethylacetate/n-hexane between aqueous acetic acid and isopropylether/n-hexane, column partition chromatography on "Celite" columns containing 70% acetic acid as the stationary phase and isopropylether/n-hexane as the mobile phase, absorption chromatography of a solution in ethylacetate or acetone. No significant change of the melting point of the purified product is recorded.
In the synthetic method for preparing CDCA from cholic acid disclosed by Fieser and Rajagopalan, a form of CDCA melting at 139.degree.-142.degree. C. is finally recovered from its solution in ethylacetate by diluting this solution with a mixture of ethyl ether and a petroleum ether.
In the process disclosed by Hofmann, a crude acid is obtained after Wolff-Kishner reduction of the corresponding ketoacid and extraction with ether-benzene or saponification of the methyl ester of CDCA and extraction with ether. When this crude acid is dissolved in a small amount of hot ethylacetate and the solution is allowed to cool, a gel is formed which after working up yields a form of CDCA having a melting point of 133.degree.-140.degree. C. Twice its volume of hot heptane is added to the hot ethylacetate solution, and, upon cooling, CDCA is obtained in form of crystalline needles having a melting point of 119.degree. C.
From The Lancet 1974, 1518, and U.S. Pat. Nos. 4,014,908 and 4,072,695, it is known that the product obtained by Hofmann is an inclusion complex wherein heptane is encaptured in the crystalline structure, and that similar needle-like crystalline inclusion complexes are obtained also in other solvent systems comprising ethylacetate-cycloalkanes and ethylacetate-alkanes. From such solvent systems the cycloalkane or the alkane are incorporated into the crystalline structure. Ethylacetate alone yields an inclusion complex with ethylacetate as the included compound. According to U.S. Pat. Nos. 4,014,908 and 4,072,695, a substantially pure solvent-free form of CDCA having a melting point of between 142.degree.-145.degree. C. can be obtained from the CDCA-solvent inclusion complex by dissolving the crystalline complex in methanol and evaporating to dryness, or in case of CDCA-cycloalkane complex by subjecting the crystals to drying in an oil pump vacuum at a pressure of 2 mm Hg and a temperature of 90.degree. C. (see col. 4, lines 13-37, and Example VII), or by dissolving the crystals in an aqueous alkaline solution and re-precipitating the CDCA from the alkaline solution by addition of a mineral acid.
Furthermore, The Lancet (loc. cit.) reported the formation of a high melting form of CDCA upon heating CDCA up to temperatures above 145.degree. C. By using differential thermal analysis the following transitions were observed with the needle-like crystalline product: an endothermic process occurs at about 120.degree. C. and is the result of release of co-crystallized solvent. This is followed by another endothermic reaction at 142.degree.-145.degree. C. which is in agreement with the melting point of one crystalline form. Increasing temperature leads to an exothermic reaction between 145.degree. C. and 160.degree. C. corresponding with recrystallization, which is finally followed by a sharp endothermic transition at 168.degree. C., corresponding with the melting-point of the high-melting form of CDCA. Yet, in The Lancet, no additional physical characteristics of the high melting form of CDCA and no methods for obtaining this high melting form of CDCA are given and no methods for preparing the high melting form of CDCA on a larger scale are suggested.
U.S. Pat. No. 4,022,806 discloses a method by which highly purified forms of CDCA having a low melting point (i.e. an amorphous form or a CDCA/solvent inclusion complex) can be transformed into a high melting form of CDCA having a melting point of about 166.degree. C. This method comprises preparing an aqueous suspension of the low melting form of CDCA, seeding the suspension with crystalline high melting CDCA material, treating the suspension at a temperature not greater than 85.degree. C. to convert the CDCA completely into material of the high melting form (see column 3, lines 28-34, and 59-68). The treatment comprises heating the aqueous suspension to a temperature not greater than 85.degree. C. with or without subjecting it to ultrasonic vibration (see column 4, lines 24-28). This process has various disadvantages. Firstly, a high-melting crystalline seeding material is required which has to be prepared separately. Furthermore, in order to effectively carry out the method of Frost et al, highly purified low melting CDCA starting material must be used and it is advisable to first prepare the crystalline calcium salt of the low melting CDCA, taking the salt up in acetic acid to form a solution of CDCA, precipitating the CDCA therefrom by dilution with water, seeding the resulting aqueous suspension of CDCA with crystalline high melting CDCA material and treating it as described above (see column 3, lines 68 to column 4, line 30).
The German Offenlegungsschrift No. 26 13 346 discloses a process for preparing a crystalline high melting CDCA material by recrystallizing raw CDCA from acetonitrile. Recrystallization from a solution in acetonitrile has also been proposed by Frost et al as a means for preparing the high melting crystalline CDCA seeding material. Even though a high melting form of CDCA can be obtained by recrystallization from acetonitrile, the use of this solvent is highly undesirable for preparing CDCA for therapeutical purposes because of the well-known toxicity of acetonitrile. The need for handling large amounts of a toxic solvent during the process naturally provides a severe disadvantage. Furthermore, in view of the well-known tendency of CDCA to retain in its crystalline structure solvents from the solution from which it is crystallized, there is the danger that at least minor amounts of the toxic acetonitrile may be retained in the high-melting crystalline material. This, of course, constitutes a potential health hazard, in particular in view of the fact that any therapeutic treatment with CDCA will usually involve administration of CDCA over a prolonged period of time.